In vivo tracking transplanted cardiomyocytes derived from human induced pluripotent stem cells using nuclear medicine imaging

Introduction Transplantation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is a promising treatment for heart failure. Information on long-term cell engraftment after transplantation is clinically important. However, clinically applicable evaluation methods have not yet been established. Methods In this study, to noninvasively assess transplanted cell engraftment, human SLC5A5, which encodes a sodium/iodide symporter (NIS) that transports radioactive tracers such as 125I, 18F-tetrafluoroborate (TFB), and 99mTc-pertechnetate (99mTcO4−), was transduced into human induced pluripotent stem cells (iPSCs), and nuclear medicine imaging was used to track engrafted human iPSC-CMs. Results To evaluate the pluripotency of NIS-expressing human iPSCs, they were subcutaneously transplanted into immunodeficient rats. Teratomas were detected by 99mTcO4− single photon emission computed tomography (SPECT/CT) imaging. NIS expression and the uptake ability of 125I were maintained in purified human iPSC-CMs. NIS-expressing human iPSC-CMs transplanted into immunodeficient rats could be detected over time using 99mTcO4− SPECT/CT imaging. Unexpectedly, NIS expression affected cell proliferation of human iPSCs and iPSC-derived cells. Discussion Such functionally designed iPSC-CMs have potential clinical applications as a noninvasive method of grafted cell evaluation, but further studies are needed to determine the effects of NIS transduction on cellular characteristics and functions.

Introduction: Transplantation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is a promising treatment for heart failure.Information on long-term cell engraftment after transplantation is clinically important.However, clinically applicable evaluation methods have not yet been established.Methods: In this study, to noninvasively assess transplanted cell engraftment, human SLC5A5, which encodes a sodium/iodide symporter (NIS) that transports radioactive tracers such as 125 I, 18 F-tetrafluoroborate (TFB), and 99m Tc-pertechnetate ( 99m TcO 4

−
), was transduced into human induced pluripotent stem cells (iPSCs), and nuclear medicine imaging was used to track engrafted human iPSC-CMs.Results: To evaluate the pluripotency of NIS-expressing human iPSCs, they were subcutaneously transplanted into immunodeficient rats.Teratomas were detected by 99m TcO 4 − single photon emission computed tomography (SPECT/CT) imaging.
NIS expression and the uptake ability of 125 I were maintained in purified human iPSC-CMs.NIS-expressing human iPSC-CMs transplanted into immunodeficient rats could be detected over time using 99m TcO 4 − SPECT/CT imaging.Unexpectedly, NIS expression affected cell proliferation of human iPSCs and iPSC-derived cells.Discussion: Such functionally designed iPSC-CMs have potential clinical applications as a noninvasive method of grafted cell evaluation, but further studies are needed to determine the effects of NIS transduction on cellular characteristics and functions.

Introduction
The number of patients with heart failure has become a global problem as the population ages, and the number of patients with atherosclerotic disease has increased due to saturated diets (1).Heart failure occurs when the heart cannot supply the blood necessary for physical activity due to myocardial cell loss and fibrosis.Advances in drug and device therapies for heart diseases have improved the prognosis of patients with heart failure (2).However, the patient number is still increasing.One reason might be the lack of a fundamental solution for cardiomyocyte loss.
In recent years, transplantation of cardiomyocytes derived from human induced pluripotent stem cells (iPSCs) has shown promise in replenishing lost cardiomyocytes and, a few clinical trials are ongoing (3,4).However, no clinical method has been established to confirm the engraftment and viability of transplanted human iPSC-derived cardiomyocytes (iPSC-CMs).The inability to assess transplanted cardiomyocyte viability means that information is lacking to determine treatment efficacy, the need for retransplantation, and the need for continued immunosuppressive agents, and establishing a non-invasive method to assess cell viability in vivo is a critical challenge to the success of cardiac regenerative therapy.
Magnetic resonance imaging (MRI) using superparamagnetic iron oxide (SPIO) and bioluminescence imaging (BLI) using the luciferin-luciferase reaction have been experimentally employed to evaluate the engraftment of transplanted cells.When SPIOincorporated cells are transplanted and survival is evaluated by MRI, it is difficult to evaluate long-term cell viability because SPIO accumulated in macrophages that have phagocytosed the transplanted dead cells is detected by MRI (5,6).BLI is often used in cell transplantation experiments with small animals.However, the problem of detection depth poses a barrier to its application in large animals, including human (7)(8)(9).In addition, since luciferase is a non-human-derived protein, the administration of luciferase-expressing cells can induce an immune response.In contrast, nuclear medicine imaging offers superior detection depth and spatial resolution compared with BLI (10).Templin et al. transduced a sodium/iodide symporter (NIS), an ion pump highly expressed in the thyroid and stomach that actively transports iodide into human iPSCs (11,12).First, NIS-expressing human iPSCs were injected into infarcted piglet hearts treated with immunosuppressants.Next, 123 I, a radioactive tracer that is taken up intracellularly via NIS, was injected into the coronary arteries, and the transplanted NIS-expressing human iPSC-derived endothelial cells were detected using single photon emission computed tomography/computed tomography (SPECT/CT).Ostrominski et al. also transduced NIS into rhesus iPSCs and transplanted cardiomyocytes derived from the NISexpressing rhesus iPSCs into immunodeficient mice (13).Subsequently, 18 F-tetrafluoroborate (TFB), a radioactive tracer taken up intracellularly via NIS (14), was administered intravenously, and the transplanted NIS-expressing rhesus iPSC-CMs were successfully detected using positron emission tomography/computed tomography (PET/CT).NIS is expressed in the thyroid and stomach and is not immunogenic.In addition, nuclear medicine imaging is already used in clinical practice, and the hurdles to its clinical application are likely to be lower than for other methods.However, there are no reports of studies evaluating the engraftment of human iPSC-CMs using nuclear medicine imaging, and further investigation using human iPSC-CMs is needed for clinical application.
In this study, we investigated whether transplanted NISexpressing human iPSC-CMs could be tracked over time using 99m Tc-pertechnetate ( 99m TcO 4 − ) SPECT/CT, which is more accessible than PET/CT.In addition, the effects of NIS transduction on cellular characteristics were investigated.

2.3.
In vitro radioactive tracer uptake study 18 F-TFB was synthesized in-house as previously described (16).The radiochemical purity of 18 F-TFB was consistently > 98%, with activity > 1.8 GBq/ml. 125I was supplied as Na 125 I (Cat.#.NEZ033l; PerkinElmer, Inc., Boston, MA, United States).The radiochemical purity of the radiolabeled compounds was greater than 99%.F-TFB and 125 I uptake study using human iPSCs were performed as previously described (17) with minor modifications.In brief, human iPSCs were seeded on iMatrix-511-coated 24-well plates at 1 × 10 5 cells/well and maintained in StemFit AK02N for 3 days.The cells were then washed twice with Hanks' balanced salt solution (HBSS, Cat.#.14175079, Thermo Fisher Scientific), and incubated in 450 µl of HBSS with or without the NIS inhibitor NaClO 4 (200 µM).2.0 MBq/ml 18 F-TFB and 0.8 MBq/ml 125 I in 50 µl of HBSS were added to each well (n = 3), followed by incubation for 60 min (37°C, 5% CO 2 ).The incubation buffer was removed, and 18 F-TFB and 125 I uptake into the cells was immediately terminated by adding 1 ml of ice-cold HBSS.
The cells were washed twice with 1 ml of ice-cold HBSS and lysed with NaOH (0.5 N, 0.5 ml) for 60 min for protein and radioactivity measurements.The protein content of each cell lysate was measured using Pierce BCA Protein Assay Kit (Cat.#.23225, Thermo Fisher Scientific) and a filter-based microplate photometer (Multiskan FC, Thermo Fisher Scientific).The radioactivity of each lysate was measured using a γ-counter (AccuFLEX γ7001, Nippon RayTech Co., Tokyo, Japan).
Following decay correction of the cell lysate, the percentage of cell uptake radioactivity in each lysate was calculated as follows: (protein volume in cell lysate (mg))] Radioactivity in added radiotracer (cpm)

Animal experiments
Animal experiments followed the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines.All mice and rats were bred under appropriate care at Department of Animal Resources, Advanced Science Research Center, Okayama University.Isoflurane (Isoflurane Inhalation Solution, Pfizer Japan, Tokyo, Japan) was used for inhalation anesthesia.Carbon dioxide inhalation was used for euthanasia.The humane endpoint for discontinuation of the experiment was defined as a decrease in body weight of more than 20%/3 days, a decrease in water and food intake, or a decrease in locomotion, when the animal's distress was deemed intolerable.No corresponding signs were observed in this study.No randomization, no strategy used to minimize potential confounders, or no blinding were done in this study.

Teratoma formation assay
To evaluate teratoma formation capacity, 1 × 10 7 NISexpressing or NIS non-expressing human iPSCs suspended in 50% Matrigel (Cat.#.354277, Corning)/50% StemFit AK02N with 10 µmol/L Y-27632 were injected subcutaneously into the back skin of 8-week-old male NSG mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ, Jackson Laboratory, Yokohama, Japan).Cell injection was performed after an acclimation period of at least one week.Six mice were used in this study.Six weeks later, teratomas were harvested after euthanasia by carbon dioxide inhalation, fixed in 10% formalin for 48 h, embedded in paraffin, and sectioned for tissue evaluation using hematoxylin and eosin staining.
To detect teratomas by SPECT/CT imaging, 1 × 10 7 human iPSCs suspended in 50% Matrigel/50% StemFit AK02N with 10 µmol/L Y-27632 were injected subcutaneously into the back skin of 8-week-old male severe combined immunodeficiency (SCID) rats (F344-II2rg/Rag2 em1lexas , NBRP-Rat with support in part by the National BioResource Project of the MEXT, Japan).Cell injection was performed after an acclimation period of at least one week.Two rats were used in this study.Six weeks later, 99m TcO 4 − SPECT/CT was performed, and teratomas were harvested and evaluated using hematoxylin and eosin staining and immunostaining.

Evaluation of human induced pluripotent stem cell-derived cardiomyocyte proliferation/viability
Human iPSC-CMs were seeded at 2 × 10 4 /well on iMatrix-511coated 96-well plates and cultured with DMEM, low glucose supplemented with 5% FBS and sodium iodide for 4 days.Cell Counting Kit-8 assay (Cat.#.CK04, Dojindo Laboratories) was used to evaluate cell numbers.Absorbance was measured using a FlexStation 3 (Molecular Devices, Sunnyvale, CA, United States).Cell area fraction was measured by ImageJ (18).

Transplantation of human induced pluripotent stem cell-derived cardiomyocytes
After an acclimatization period of at least one week, the inguinal skin of 8-week-old male SCID rats (F344-II2rg/Rag2 em1lexa ) was incised under isoflurane (Isoflurane Inhalation Solution, Pfizer Japan, Tokyo, Japan) inhalation anesthesia.Human iPSC-CMs at 1 × 10 7 were suspended in MatriMix (511) with 10 µmol/L Y-27632 (Cat.#. 899001, Nippi, Tokyo, Japan) and injected into the inguinal subcutaneous adipose tissue.99m TcO 4 − SPECT/CT was performed 1, 6, and 10 weeks after cell injection.After an acclimatization period of at least one week, 10-week-old male SCID rats were intubated and ventilated, and the left fourth intercostal space was opened under isoflurane inhalation anesthesia.A 7 mm-diameter aluminum rod cooled in liquid nitrogen for 1 min was placed once on the left ventricle to create cryoinjury, and 1 × 10 7 human iPSC-CMs suspended in MatriMix (511) with 10 µmol/L Y-27632 (total volume 100 µl) were then injected into three locations around the injury.99m TcO 4 − SPECT/ CT was performed 12 and 16 weeks after cell injection.

Detection of transplanted cells using 99m
TcO 4 − SPECT/CT SPECT/CT scans were performed using a dedicated smallanimal SPECT/CT system (NanoSPECT/CT, Mediso Medical Imaging Systems, Budapest, Hungary).General anesthesia was induced by using 5% isoflurane and maintained with 2% isoflurane during the experiment.The rats were injected 140-150 MBq of 99m TcO 4 − via tail vein.The acquisition started at 30 min post-injection with a total scan time of 40 min.Following the SPECT imaging session, CT was conducted for 10 min to confirm the precise position of the injected sites.Fusion images of CT and SPECT were created, and the obtained images were analyzed using the public domain tool such as AMIDE imaging software (A Medical Imaging Data Examiner) (21).99m TcO 4 − uptake was calculated by multiplying the volume of regions of interest with increased 99m TcO 4 − uptake (mm 3 ) and its mean uptake value and normalizing with both the injected dose (MBq) and body weight (g) of the rat.The uptake of radioactive tracer was quantitatively evaluated in four rats for which SPECT/CT could be performed over time.
Eight rats (inguinal injection, 1; heart injection, 7) that died before SPECT/CT imaging, 5 rats (inguinal injection, 1; heart injection, 4) that died during SPECT/CT imaging, and 1 rat (inguinal injection) that died after the first SPECT/CT imaging were excluded from the analysis.

Statistical analysis
All data are expressed as means ± standard deviation (SD).Statistical analysis was performed by Student's t-test using SPSS Statistics 24 (IBM, Armonk, NY, United States).Statistical significance was set at p < 0.05.

Generation of SLC5A5 transgenic human iPSC lines
To prevent transgene silencing during iPSC differentiation, the SLC5A5 gene encoding NIS was transduced into the AAVS1 locus known as a safe harbor (22-24) using TALEN (Figure 1A).EGFP and SLC5A5 were transduced together to determine the presence or absence of NIS expression using fluorescence microscopy.Gene knock-in to the AAVS1 locus was confirmed using PCR, and human iPSC lines with transgenes in both alleles were used in subsequent experiments: NIS-expressing human iPSC lines, NIS (+) #4, #6 and #8; NIS non-expressing human iPSC lines, NIS (−) #1 and #2 (Figures 1B,C).Fluorescence microscopy confirmed EGFP expression in the transgenic human iPSC lines (Figure 1D).

Expression of functional NIS in human iPSCs
Undifferentiated markers of NIS-expressing human iPSCs and NIS non-expressing human iPSCs were confirmed by immunostaining.NIS protein expression was confirmed by immunostaining, and the signal was detected only in NISexpressing human iPSCs (Figure 2A).In vitro radioactive tracer uptake experiments were performed to reveal the functionality of the expressed NIS. NIS-expressing human iPSCs acquired the ability to take up 125 I and 18 F-TFB.This uptake was completely inhibited by NaClO 4 , an inhibitor of NIS, confirming that the uptake of these radioactive tracers was due to the transduced NIS (Figures 2B,C).NIS-expressing human iPSCs and NIS non-expressing human iPSCs were transplanted subcutaneously into the back of immunodeficient mice and observed 6 weeks later.NIS nonexpressing human iPSC-injected mice showed grossly visible mass formation.In contrast, mice injected with NIS-expressing human iPSCs showed no obvious mass formation on the external surface (Figure 3A).When the skin at the injection site was cut open, a small mass was observed in NIS-expressing human iPSCinjected mice, and histological analysis confirmed that it was a teratoma (Figure 3B).
Next, NIS-expressing human iPSCs were injected into the right side of the back and NIS non-expressing human iPSCs into the left side of the back of immunodeficient rats, respectively, and 99m TcO 4 − SPECT/CT was performed 6 weeks later.As with transplantation into mice, NIS-expressing human iPSC-derived teratomas were smaller than NIS non-expressing human iPSCderived teratomas, but uptake of radioactive tracer was observed only on the right side of the back (Figure 3C).Histological analysis confirmed that NIS-expressing human cells formed teratomas (Figure 3D).

Effect of NIS overexpression on cell proliferation in human iPSCs
The reason why overexpression of NIS reduces the size of teratomas was investigated in vitro.NIS-expressing human iPSCs grew well in Essential 8 Flex Medium, the maintenance iPSC medium.However, addition of 30 µmol/L sodium iodide (NaI), the substrate of NIS, for 4 days significantly suppressed cell proliferation (Figures 4A,B).This concentration of iodine is approximately 30 times the physiological blood concentration and thus cannot explain the phenomenon seen in teratomas.In contrast, when iPSCs were cultured for 4 days with RPMI 1640/ B-27 supplement medium, a commonly used medium for differentiation, NIS-expressing iPSC-derived differentiated cells showed a marked decrease in proliferation.In addition, the addition of NaI rescued the inhibition of their cell proliferation (Figures 4C-E).

Characteristics of NIS-expressing human iPSC-CMs
Cardiomyocytes were induced from NIS-expressing human iPSCs and NIS non-expressing human iPSCs.After purification with no glucose DMEM supplemented with lactic acid, most cells expressed NIS (Figure 5A), working cardiomyocyte markers (NKX2-5 and Connexin 43), pan-cardiomyocyte markers (cardiac Troponin T, and α-actinin), and ventricular cardiomyocyte marker (MLC2v) (Figures 5B-D).NIS-expressing human iPSC-CMs were capable of 125 I uptake, which was inhibited by the NIS inhibitor NaClO 4 (Figure 5E).The effect of iodine on cell proliferation was examined.The addition of 0.3 and 3 µmol/ L NaI for 10 days significantly increased the number of NISexpressing human iPSC-CMs compared to NIS non-expressing human iPSC-CMs (Figure 5F).In contrast, no effect of NaI on cell number was observed in NIS non-expressing human iPSC-CMs (Supplementary Figures S1A,B).− SPECT/CT was performed one week later.Radioactive tracer uptake was detected only in the right inguinal subcutaneous adipose tissue (Figure 6A).Histologically, human cardiomyocytes were detected in the subcutaneous adipose tissue of the bilateral inguinal regions (Figure 6B).

3.7.
In vivo tracking transplanted NIS-expressing human iPSC-CMs using 99m TcO 4 − SPECT/CT NIS-expressing human iPSC-CMs were injected into the right inguinal subcutaneous adipose tissue of immunodeficient rats, and 99m TcO 4 − SPECT/CT was performed 6 and 10 weeks after transplantation, showing uptake of radioactive tracer at the transplant site over time (Figures 7A,B).NIS-expressing human iPSC-CMs were injected into the heart, and 99m TcO 4 − SPECT/CT showed uptake of radioactive tracer in the heart over time (Figure 7C).The 99m Tc signal was measured and the uptake value was assessed over time (Figure 7D).The same number of cardiomyocytes were administered, but the variation in 99m TcO 4 − uptake is likely to be large.In the histological evaluation, almost all of the transplanted cells were NIS-positive and cardiac troponin T-positive.Therefore, this variation appears to be more a matter of dosing technique than a possible reduction in uptake due to decreased NIS expression (Figures 7E,F).

Discussion
This study showed that NIS-transduced human iPSCs and iPSC-derived cells acquired radioactive tracer uptake ability to track iPSC-derived cells over time using 99m    mice (13).The study differed from ours by the species of iPSCs, the species of recipient animals, and the detection modalities.This study analyzed NIS-transduced human iPSCs for future clinical applications.We demonstrated that in vivo imaging using NIS transduction is possible even in human iPSC-CMs.In addition, we confirmed the viability of orthotopically or ectopically transplanted human iPSC-CMs in rats, which are larger than mice.Moreover, we used 99m TcO 4 − SPECT/CT to detect engrafted NIS-expressing human iPSC-CMs.Although SPECT/CT is less sensitive than PET/CT, it has the advantage of simultaneous multi-radionuclide imaging, which can provide information on myocardial blood flow and metabolism simultaneously (11,(25)(26)(27).In addition, it is more convenient than PET/CT due to the lower cost of radioactive tracers and its widespread use in numerous facilities.Therefore, we confirmed that 99m TcO 4 − SPECT/CT can detect transplanted human iPSC-CMs.The ability to noninvasively detect and quantify transplanted cardiomyocytes is expected to provide the information necessary to determine the efficacy of cell therapy and to decide on treatment strategies.The degree of tracer uptake showed no clear increasing or decreasing trend over time.This is similar to the report by    (8), suggesting proliferation of the transplanted cells.There are differences in the method of induction of cardiomyocytes from iPSCs between these studies, but it may be necessary to consider the effect of transgenes on cardiomyocyte proliferation in vivo.
Interestingly, the effect of NIS transduction on cell proliferation varied by the cell type.NIS-expressing human iPSCs grew as well as NIS non-expressing human iPSCs in maintenance medium, and their proliferation was not affected by physiological iodine concentrations (28).However, the addition of high concentration of NaI inhibited their growth.In contrast, the proliferation of NIS-expressing human iPSC-derived differentiated cells was inhibited when cultured in RPMI 1640/B-27 supplement medium, a commonly used basic medium for differentiation.The growth inhibition was partially rescued by adding NaI.Furthermore, human iPSC-CMs showed a slight but significant increase in cell number with the addition of NaI, corresponding to physiological iodine concentrations.Although the details of these mechanisms are unclear, it is suggested that overexpressed NIS affects the cell cycle at each differentiation stage.In addition, NIS-expressing human iPSCs formed small masses in the teratoma formation test.Histological study showed that NISexpressing human iPSC-derived small mass contained endodermal, mesodermal and ectodermal tissues as shown in Figure 3B.This means that pluripotency is maintained in NISexpressing human iPSCs.This result suggests the potential benefit of reducing the risk of tumor formation by undifferentiated cells intermingled with transplanted cells.Previous studies did not mention these characteristics (11, 13,29).Templin et al. reported that no teratoma or other obvious tumor formation was detected in pig infarcted hearts injected with NIS-expressing human iPSCs (11).Moreover, Guerrieri et al. reported that endogenous and exogenous NIS promoted apoptosis in liver cancer cell lines (30).Therefore, the effects of NIS overexpression on characteristics of human iPSCs and iPSC-CMs require further investigation.
Threre are some limitations in this study.First, this study did not adequately examine human iPSC-CM transplantation into the heart.Unfortunately, most II2rg/Rag2-knockout rats that received cell injection into the heart died intraoperatively or during SPECT/CT imaging under inhalation anesthesia, making evaluation difficult.This may be a technical problem, but our experience with II2rg/Rag2-knockout rats used in this study gave us the impression that they had a higher mortality rate during and after open heart surgery than the commonly used Wistar rats (data not shown).Compared to Il2rg-knockout rats, Il2rg/ Rag2 knockout rats are characterized by their inability to produce natural killer cells (31).We do not know if this is related to the cause of the high intraoperative mortality rate in this study.The use of F344/NJcl.Cg-Foxn1 rnu (32) and F344-Il2rg em7Kyo (33) for cell transplantation into the heart has been reported, so the use of these rats may advance research on cell transplantation study into the heart.Second, the number of cells at the detection threshold was not studied.Ostrominski et al. showed that the in vivo detection threshold using PET/CT was 1 × 10 5 NIS-expressing rhesus iPSC-CMs injected into mice.Since the sensitivity of SPECT is lower than that of PET, the detection threshold is presumed to be higher than the number of cells mentioned above.Since the number of human iPSC-CMs used in clinical trials is very large (1 × 10 8 cells) (3), it is expected that nuclear medicine imaging with NIS gene transduction will detect transplanted cardiomyocytes.Third, since the study was not performed in large animals, it is necessary to verify whether transplanted cells are detectable in large animals.However, the depth of penetration and spatial resolution of SPECT are superior to those of BLI (10,11), and since 99m TcO 4 − SPECT/CT is already in clinical use, clinical application of this method is expected.The pro-arrhythmic effect, which is a problem with iPSC-CM transplantation, need to be examined because NIS is electrogenic (34-37).No structural abnormalities or arrhythmias of the heart have been reported in transgenic mice with MHC promoter-driven NIS, which is specifically expressed in cardiomyocytes (38).Moreover, Ostrominski et al. showed little electrophysiological effect of transduced NIS in the range of physiological iodine concentration in monkey iPSC-CMs in vitro (13).However, to examine the pro-arrhythmic effect in vivo, transplantation experiments with large animals are necessary because rodent heart rates are too high to assess arrhythmias induced by transplanted human iPSC-CMs.In summary, NIS-expressing human iPSC lines were established, and NIS-expressing human cardiomyocytes were induced.Transplanted NIS-expressing human iPSC-derived cells were detected using 99m TcO 4 − SPECT/CT.99m TcO 4 − SPECT/CT has already been used in clinical practice, and this approach is expected to be applied clinically to confirm the viability and engraftment of transplanted human iPSC-CMs.The effects of NIS overexpression on human iPSC and iPSC-CM characteristics and functions must be studied extensively.
The animal study was approved by the Animal Care and Use Committee, Okayama University.The study was conducted in accordance with the local legislation and institutional requirements.

FIGURE 1
FIGURE 1 Generation of SLC5A5 transgenic human induced pluripotent stem cell (iPSC) lines.(A) Transduction of human SLC5A5 gene to AAVS1 locus in human induced pluripotent stem cells (iPSCs) using TALEN.(B) The difference in size of polymerase chain reaction (PCR) product depends on the presence or absence of the transgene.(C) Determination of the presence or absence of gene transfer to the AAVS1 locus using PCR of DNA extracted from each iPSC line.(D) EGFP expression in each human iPSC line.Scale bars are 200 µm.

3. 6 . 4 −
In vivo detection of transplanted NIS-expressing human iPSC-CMs by 99m TcO SPECT/CT NIS-expressing human iPSC-CMs were injected into the right inguinal subcutaneous adipose tissue and NIS non-expressing iPSC-CMs into the left inguinal subcutaneous adipose tissue of an immunodeficient rat, and 99m TcO 4

FIGURE 4 NIS
FIGURE 4 NIS-expressing human induced pluripotent stem cell (iPSC) lines showed slower proliferation in differentiation medium than NIS non-expressing human iPSC lines did.Sodium iodide (NaI) rescued poor proliferation of NIS-expressing cells.(A) NIS-expressing human iPSCs grew as well as NIS non-expressing human iPSCs in Essential 8 Flex Medium (maintenance medium for human pluripotent stem cells).Cells were cultured in Essential 8 Flex Medium for 4 days with or without NaI, and nuclei (blue) and F-actin (red) were stained.Scale bars are 1 mm.(B) Cell area/surface area was measured (N = 3 in each).*p < 0.001 (ANOVA/Bonferroni).(C) Cells were cultured with RPMI/B27 medium (basal medium for differentiation) for 4 days with or without NaI, and nuclei (blue) and F-actin (red) were stained.Scale bars are 1 mm.(D) Cell area/surface area was measured (N = 3 in each, Student-t test).(E) Cell counts were evaluated by MTT assay (N = 6 in each).*p < 0.01, **p < 0.001 vs. NIS (+) w/o NaI (ANOVA/Bonferroni).

FIGURE 5
FIGURE 5 Induction of NIS-expressing human pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).(A) Immunostaining of NIS in NIS-expressing and NIS nonexpressing human iPSC-CMs at differentiation day 31.Scale bars are 400 µm.(B) Immunostaining of NKX2-5, a marker of working cardiomyocytes, and cardiac troponin T (cTnT) in NIS-expressing and NIS non-expressing human iPSC-CMs at differentiation day 31.Scale bars are 200 µm.(C) Immunostaining of α-actinin and Connexin 43 in NIS-expressing and NIS non-expressing human iPSC-CMs.Scale bars are 100 µm.(D) Immunostaining of α-actinin and MLC2v, a marker of ventricular cardiomyocytes, on differentiation day 50 in NIS-expressing and NIS non-expressing human iPSC-CMs.Scale bars are 100 µm.(E) Uptake of 125 I of human iPSC-CMs at differentiation day 35 in vitro.NaClO 4 is an inhibitor of NIS.Data are shown as mean ± standard deviation (n = 5 in each).*p < 0.001 vs. NIS (+) w/o 200 µM NaClO 4 (ANOVA/Bonferroni).(F) NIS-expressing human iPSC-CMs were cultured for 10 days from differentiation day 21, and cell viability was evaluated using CCK-8 assay.Data are shown as mean ± standard deviation (n = 6 in each, ANOVA/Bonferroni).

FIGURE 6
FIGURE 6 Detection of transplanted human pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).(A) NIS-expressing human iPSC-CMs were injected into the right inguinal subcutaneous adipose tissue and NIS non-expressing human iPSC-CMs into the left inguinal subcutaneous adipose tissue in an immunodeficient rat.One week later, 99m TcO 4 − SPECT/CT was performed to detect NIS-expressing human iPSC-CMs (surrounded by a white dashed line).(B) Engraftment of both NIS-expressing human iPSC-CMs and NIS non-expressing human iPSC-CMs were histologically confirmed by immunostaining of human Ku80, NIS and cardiac troponin T (cTnT).Scale bars are 200 µm.

FIGURE 7 Long
FIGURE 7 Long-term tracking transplanted human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).(A) Six weeks after injection of NISexpressing human iPSC-CMs into the right inguinal subcutaneous adipose tissue of an immunodeficient rat, 99m TcO 4 − SPECT/CT was performed to detect the injected cells (surrounded by a white dashed line).(B) 99m TcO 4 − SPECT/CT image of the same rat shown in (A) 10 weeks after cell injection.Tracer uptake was observed in the right inguinal subcutaneous adipose tissue (surrounded by a white dashed line).(C) Sixteen weeks after injection of NIS-expressing iPSC-CMs into the cryoinjured heart of an immunodeficient rat, 99m TcO 4 − SPECT/CT was performed to detect the injected cells (surrounded by a white dashed line).(D) Quantification of 99m Tc signals from transplanted cells over time.(E) Engraftment of NIS-expressing human iPSC-CMs was histologically confirmed by immunostaining of human Ku80, NIS, cardiac troponin T (cTnT), and MLC2v.Scale bars are 100 µm.(F) Engraftment of NIS-expressing human iPSC-CMs was histologically confirmed by immunostaining of human Ku80, NIS and cTnT.Scale bars are 100 µm.
(13)ominski et al. who detected NIS-expressing rhesus iPSC-CMs by PET/CT(13).In contrast, Funakoshi et al. detected luciferaseexpressing human iPSC-CMs by BLI and found that the signal intensity remained enhanced from 2 weeks to 3 months after transplantation